Lipophilic benzothiophenes

ABSTRACT

The invention provides novel benzothiophenes of the formula (I): ##STR1## wherein R 1  is N-pyrrolidinyl or N-piperidinyl; R 2  and R 3  are independently hydrogen,--CO--(C 10  -C 22  alkyl), --CO--(C 10  -C 22  branched alkyl), --CO--(C 10  -C 22  alkenyl), --CO--(C 10  -C 22  polyalkenyl), --CO--(C 10  -C 22  alkynyl),or --CO--(CH 2 ) n  COR 4  ; provided R 2  and R 3  are not both dodecanoyl, and one of R 2  or R 3  is not hydrogen 
     R 4  is -3-cholesteryl or --O(CH 2 ) 2  (OR 5 )CH 2  OR 6  ; 
     R 5  and R 6  are independently hydrogen, --CO--(C 10  -C 22  alkyl), --CO--(C 10  -C 22  branched alkyl), --CO--(C 10  -C 22  alkenyl), --CO--(C 10  -C 22  polyalkenyl), or --CO--(C 10  -C 22  alkynyl); provided one of R 5  or R 6  is not hydrogen; 
     n is 0-4; and pharmaceutically acceptable salts and solvates thereof. 
     The present invention further provides pharmaceutical compositions containing compounds of formula I, and the use of such compounds.

This application is a division of application Ser. No. 08/943,749, filedOct. 3, 1997, now U.S. Pat. No. 5,929,092, which is a division of Ser.No. 08/707,680 filed Sep. 4, 1996 U.S. Pat. No. 5,726,168 which claimsthe benefit of U.S. Provisional Application No. 60/005,140 filed Oct.12, 1995.

BACKGROUND OF THE INVENTION

There are three types of lesions found in the arteries which areassociated with atherosclerosis: fatty streaks, fibrous plaques, andcomplicated plaques. Fatty streaks occur early in life and consist of anaccumulation of lipid-filled macrophages and smooth muscle cells (foamcells) and accumulated fibrous tissue on the intima. In general, thesefatty streaks appear not to be particularly dangerous in themselves;however, they may be contributory to the formation of fibrous plaques.Fibrous plaques are raised lesions on the intima. These plaques consistof a central core of extracellular lipid and necrotic cell debris andare covered with an overlayment of smooth muscle cells and collagenmatrix. This makes the fibrous plaque foci, a place of constricted bloodflow in the artery. The fibrous plaque is characteristic of advancingatherosclerotic disease. The complicated plaque is a calcified fibrousplaque and is an area of thrombosis, necrosis, and ulceration. Thisplaque constricts the blood flow and causes stenosis that can lead toorgan insufficiency. The site of a complicated plaque is, also, an areaof weakened arterial wall which may fail, causing aneurysm formation andhemorrhaging.

One theory of atherogensis is the reaction-to-injury hypothesis.According to this hypothesis, the lining endothelial cells of the arteryare exposed to acute, repeated acute, or chronic injury, which causesthe cells to detach from one another, thus exposing the underlyingconnective tissue bed. This break in the continuous system ofendothelium elicits platelet adhesion, aggregation, and the formation ofmicrothrombi. This platelet interaction causes the release of mitogenicfactors leading to the proliferation of smooth muscle cells, theproduction of matrix, and the trapping of lipids from the serum.Although this repairs the immediate break in the system, the disturbancein the blood around the lesion often causes further damage toendothelium in adjacent areas, especially down stream from the initialinsult, thus increasing the plaque size. This process continues to buildthe lesion and leads to constriction of the blood flow and eventualocclusion or failure of the arterial wall.

Today, balloon angioplasty is one of the most common procedures used intreating atherosclerotic plaques, especially for relatively smallplaques. It is often preferred over by-pass surgery, in that it is lessexpensive and is a great deal less traumatic to the patient.

Although angioplasty is very effective at initially opening the occludedartery, this artery often fails to remain open for an extended period oftime. Within one year, 30-50% of the arteries opened by angioplasticsurgery are occluded in the same or adjacent location as the initialblockage. The process by which this re-occlusion occurs is calledrestenosis. For reviews covering the morphologic changes andbiopathology of restenosis following angioplasty see: Haudenschild, C.C., Am. J. of Med., 94, (Suppl 4A), p. 4A-40S-4A44S (1993) and Waller,B. F., et al. Radiology, 174, (3), p. 961-967 (1990).

Currently, there is no treatment for the restenosis of an artery, otherthan repeating the angioplasty, which may exacerbate the problem, orperforming a more extensive procedure such as by-pass surgery.

It has been shown that another benzothiophene, raloxifene (formula I, R₁is n-piperidenyl, and R₂ and R₃ are hydrogen) is active in experimentalmodels in inhibiting restenosis (see: EP652,003, published May 10,1995). In experimental models conducted in vivo, raloxifene wasadministered via a systemic route (oral) demonstrating its beneficialeffect at inhibiting restenosis. Additionally, it has now been shownthat raloxifene is capable of inhibiting intimal thickening at a localsite of angioplasty insult. This result is of great significance in thatraloxifene is of a chemical class of compounds known as mixed estrogenagonist/antagonists, or SERMs, selective estrogen receptor modulators.Administration of a compound having a similar profile as that ofraloxifene, but at the local site, would be an advantage for thetreatment of restenosis induced by angioplastic intervention.

Ideally, it would be desirable to administer such an agent directly intothe plaque at the time of angioplasty. This is problematic in thatraloxifene is not very soluble in the highly lipophilic matrix found inthe atherosclerotic plaque. Additionally, large volumes of solvent wouldbe necessary to dissolve an effective amount of raloxifene which wouldthen have to be delivered via the angioplasty catheter into theatherosclerotic plaque. Both of these raise practical concerns over theuse of either raloxifene or its known derivatives from use as locallyactive agents for preventing restenosis via an angioplasty catheter.

It would be of great benefit if there were an efficient non-surgicaltreatment for restenosis. It would be of particular benefit if such atreatment could be confined to the immediate locality of the occludingplaque, since this would limit any potential side-effects of thetreatment. A treatment such as a drug which could be delivered locallyto the plaque site at the time of angioplasty and prevent restenosis atthat site would be ideal.

"Post-menopausal syndrome" is a term used to describe variouspathological conditions which frequently affect women who have enteredinto or completed the physiological metamorphosis known as menopause.Although numerous pathologies are contemplated by the use of this term,three major effects of post-menopausal syndrome are the source of thegreatest long-term medical concern: osteoporosis, cardiovascular effectssuch as hyperlipidemia, and estrogen-dependent cancer, particularlybreast and uterine cancer.

Osteoporosis describes a group of diseases which arise from diverseetiologies, but which are characterized by the net loss of bone mass perunit volume. The consequence of this loss of bone mass and resultingbone fracture is the failure of the skeleton to provide adequatestructural support for the body. One of the most common types ofosteoporosis is that associated with menopause. Most women lose fromabout 20% to about 60% of the bone mass in the trabecular compartment ofthe bone within 3 to 6 years after the cessation of mensus. This rapidloss is generally associated with an increase of bone resorption andformation. However, the resorptive cycle is more dominant and the resultis a net loss of bone mass. Osteoporosis is a common and serious diseaseamong post-menopausal women.

There are an estimated 25 million women in the United States, alone, whoare afflicted with this disease. The results of osteoporosis arepersonally harmful and also account for a large economic loss due itschronicity and the need for extensive and long term support(hospitalization and nursing home care) from the disease sequelae. Thisis especially true in more elderly patients. Additionally, althoughosteoporosis is not generally thought of as a life threateningcondition, a 20% to 30% mortality rate is related with hip fractures inelderly women. A large percentage of this mortality rate can be directlyassociated with post-menopausal osteoporosis.

The most vulnerable tissue in the bone to the effects of post-menopausalosteoporosis is the trabecular bone. This tissue is often referred to asspongy or cancellous bone and is particularly concentrated near the endsof the bone (near the joints) and in the vertebrae of the spine. Thetrabecular tissue is characterized by small osteoid structures whichinter-connect with each other, as well as the more solid and densecortical tissue which makes up the outer surface and central shaft ofthe bone. This inter-connected network of trabeculae gives lateralsupport to the outer cortical structure and is critical to thebio-mechanical strength of the overall structure. In post-menopausalosteoporosis, it is, primarily, the net resorption and loss of thetrabeculae which leads to the failure and fracture of bone. In light ofthe loss of the trabeculae in post-menopausal women, it is notsurprising that the most common fractures are those associated withbones which are highly dependent on trabecular support, e.g., thevertebrae, the neck of the weight bearing bones such as the femur andthe fore-arm. Indeed, hip fracture, collies fractures, and vertebralcrush fractures are hall-marks of post-menopausal osteoporosis.

At this time, the only generally accepted method for treatment ofpost-menopausal osteoporosis is estrogen replacement therapy. Althoughtherapy is generally successful, patient compliance with the therapy islow primarily because estrogen treatment frequently produces undesirableside effects.

Throughout premenopausal time, most women have less incidence ofcardiovascular disease than age-matched men. Following menopause,however, the rate of cardiovascular disease in women slowly increases tomatch the rate seen in men. This loss of protection has been linked tothe loss of estrogen and, in particular, to the loss of estrogen'sability to regulate the levels of serum lipids. The nature of estrogen'sability to regulate serum lipids is not well understood, but evidence todate indicates that estrogen can upregulate the low density lipid (LDL)receptors in the liver to remove excess cholesterol. Additionally,estrogen appears to have some effect on the biosynthesis of cholesterol,and other beneficial effects on cardiovascular health.

It has been reported in the literature that post-menopausal women havingestrogen replacement therapy have a return of serum lipid levels toconcentrations to those of the pre-menopausal state. Thus, estrogenwould appear to be a reasonable treatment for this condition. However,the side-effects of estrogen replacement therapy are not acceptable tomany women, thus limiting the use of this therapy. An ideal therapy forthis condition would be an agent which would regulate the serum lipidlevel as does estrogen, but would be devoid of the side-effects andrisks associated with estrogen therapy.

The third major pathology associated with post-menopausal syndrome isestrogen-dependent breast cancer and, to a lesser extent,estrogen-dependent cancers of other organs, particularly the uterus.Although such neoplasms are not solely limited to a post-menopausalwomen, they are more prevalent in the older, post-menopausal population.Current chemotherapy of these cancers has relied heavily on the use ofanti-estrogen compounds such as, for example, tamoxifen. Although suchmixed agonist-antagonists have beneficial effects in the treatment ofthese cancers, and the estrogenic side-effects are tolerable in acutelife-threatening situations, they are not ideal. For example, theseagents may have stimulatory effects on certain cancer cell populationsin the uterus due to their estrogenic (agonist) properties and they may,therefore, be contraproductive in some cases. A better therapy for thetreatment of these cancers would be an agent which is an anti-estrogencompound having negligible or no estrogen agonist properties onreproductive tissues.

In response to the clear need for new pharmaceutical agents which arecapable of alleviating the symptoms of, inter alia, post-menopausalsyndrome, the present invention provides new benzothiophene compounds,pharmaceutical compositions thereof, and methods of using such compoundsfor the treatment of post-menopausal syndrome and other estrogen-relatedpathological conditions such as those mentioned below.

Uterine fibrosis (uterine fibroid disease) is an old and ever presentclinical problem which goes under a variety of names, including uterinefibroid disease, uterine hypertrophy, uterine lieomyomata, myometrialhypertrophy, fibrosis uteri, and fibrotic metritis. Essentially, uterinefibrosis is a condition where there is an inappropriate deposition offibroid tissue on the wall of the uterus.

This condition is a cause of dysmenorrhea and infertility in women. Theexact cause of this condition is poorly understood but evidence suggeststhat it is an inappropriate response of fibroid tissue to estrogen. Sucha condition has been produced in rabbits by daily administrations ofestrogen for 3 months. In guinea pigs, the condition has been producedby daily administration of estrogen for four months. Further, in rats,estrogen causes similar hypertrophy.

The most common treatment of uterine fibrosis involves surgicalprocedures both costly and sometimes a source of complications such asthe formation of abdominal adhesions and infections. In some patients,initial surgery is only a temporary treatment and the fibroids regrow.In those cases a hysterectomy is performed which effectively ends thefibroids but also the reproductive life of the patient. Also,gonadotropin releasing hormone antagonists may be administered, yettheir use is tempered by the fact they can lead to osteoporosis. Thus,there exists a need for new methods for treating uterine fibrosis, andthe methods of the present invention satisfy that need.

Endometriosis is a condition of severe dysmenorrhea, which isaccompanied by severe pain, bleeding into the endometrial masses orperitoneal cavity and often leads to infertility. The cause of thesymptoms of this condition appear to be ectopic endometrial growthswhich respond inappropriately to normal hormonal control and are locatedin inappropriate tissues. Because of the inappropriate locations forendometrial growth, the tissue seems to initiate local inflammatory-likeresponses causing macrophage infiltration and a cascade of eventsleading to initiation of the painful response. The exact etiology ofthis disease is not well understood and its treatment by hormonaltherapy is diverse, poorly defined, and marked by numerous unwanted andperhaps dangerous side effects.

One of the treatments for this disease is the use of low dose estrogento suppress endometrial growth through a negative feedback effect oncentral gonadotropin release and subsequent ovarian production ofestrogen; however, it is sometimes necessary to use continuous estrogento control the symptoms. This use of estrogen can often lead toundesirable side effects and even the risk of endometrial cancer.

Another treatment consists of continuous administration of progestinswhich induces amenorrhea and by suppressing ovarian estrogen productioncan cause regressions of the endometrial growths. The use of chronicprogestin therapy is often accompanied by the unpleasant CNS sideeffects of progestins and often leads to infertility due to suppressionof ovarian function.

A third treatment consists of the administration of weak androgens,which are effective in controlling the endometriosis; however, theyinduce severe masculinizing effects. Several of these treatments forendometriosis have also been implicated in causing a mild degree of boneloss with continued therapy. Therefore, new methods of treatingendometriosis are desirable.

SUMMARY OF THE INVENTION

The invention provides novel benzothiophenes of the formula (I):##STR2## wherein R₁ is N-pyrrolidinyl or N-piperidinyl; R₂ and R₃ areindependently hydrogen, --CO--(C₁₀ -C₂₂ alkyl), --CO--(C₁₀ -C₂₂ branchedalkyl), --CO--(C₁₀ -C₂₂ alkenyl), --CO--(C₁₀ -C₂₂ polyalkenyl),--CO--(C₁₀ -C₂₂ alkynyl), or --CO--(CH₂)_(n) COR₄ ; provided R₂ and R₃are not both dodecanoyl, and one of R₂ or R₃ is not hydrogen

R₄ is -3-cholesteryl or --O(CH₂)₂ (OR₅)CH₂ OR₆ ;

R₅ and R₆ are independently hydrogen, --CO--(C₁₀ -C₂₂ alkyl), --CO--(C₁₀-C₂₂ branched alkyl), --CO--(C₁₀ -C₂₂ alkenyl), --CO--(C₁₀ -C₂₂polyalkenyl), or --CO--(C₁₀ -C₂₂ alkynyl); provided one of R₅ or R₆ isnot hydrogen;

n is 0-4; and pharmaceutically acceptable salts and solvates thereof.

Included within the scope of compounds of formula I are isomers ofasymmetric centers and cis/trans isomers associated with alkenylmoieties.

The present invention further relates to pharmaceutical compositionscontaining compounds of formula I, optionally containing estrogen orprogestin, and the use of such compounds, alone, or in combination withestrogen or progestin, for alleviating the symptoms of post-menopausalsyndrome, particularly osteoporosis, cardiovascular related pathologicalconditions, and estrogen-dependent cancer. As used herein, the term"estrogen" includes steroidal compounds having estrogenic activity suchas, for example 17β-estradiol, estrone, conjugated estrogen (Premarin®),equine estrogens, 17β-ethynyl estradiol, and the like. As used herein,the term "progestin" includes compounds having progestational activitysuch as, for example, progesterone, norethylnodrel, nongestrel,megestrol acetate, norethindrone, and the like.

Further, this invention provides for a method of administration of acompound of formula I at the site of an atherosclerotic plaque.

This invention also provides for methods of use of the compounds offormula I for the local treatment and prevention of restenosisadministered during the angioplasty of atherosclerotic plaques.

DETAILED DESCRIPTION OF THE INVENTION

The current invention relates to the discovery of a new series oflipophilic esters of 2-phenyl-3-aroylbenzo[b]thiophenes shown in formulaI. These compounds are useful for treating or preventing restenosis,particularly by administration at a local site, following angioplasty ofan atherosclerotic plaque, as well as inhibiting pathological conditionsassociated with post-menopausal syndrome.

The term "inhibit" includes its generally accepted meaning whichincludes prohibiting, preventing, restraining, and slowing, stopping, orreversing progression, severity, or a resultant symptom. As such, themethods include both medical therapeutic and/or prophylacticadministration, as appropriate.

The general chemical terms used in the description of a compound offormula I have their usual meanings. For example, the term "--CO(C₁₀₋₂₂alkyl or C₁₀ -C₂₂ branched alkyl)" would include --CO(C₁₄ -C₂₂ alkyl)and --CO(C₁₄ -C₂₂ branched alkyl), and groups such as decanoyl,undecanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, arachidoyl,2,2-dimethylundecanoyl, d,1-2-ethylundecanoyl, and the like. The term"C₁₀ -C₂₂ alkenyl or C₁₀ -C₂₂ poly-alkenyl" would include groups suchas: palmitoleoyl, oleoyl, linoleoyl, linolenoyl, arachidonoyl, and thelike, including natural and un-natural cis/trans isomers. The term "C₁₀-C₂₂ alkynyl" would include such groups as 2-alkynyl-undecanoyl,3-alkynyl-stearoyl, and the like.

The compounds of this invention may be prepared by known and/oranalogous chemical synthesis methods well known in the art. Briefly, thestarting benzothiophene, such as, raloxifene, can be prepared fromreadily available starting materials by procedures described in the U.S.Pat. Nos., 4,133,814 and 4,418,068, incorporated herein by reference.

The preparation of the acyl esters of the 4' and 6 phenolic hydroxyls ofraloxifene can be accomplished with the use of activated carboxylates ofthe long chain acids, many of which are commercially available. Examplesof such activated carboxylic acids are: stearoyl chloride, stearoylanhydride, plamitoyl chloride, arachidonoyl chloride, and the like. Theacylation reaction may be carried out in a variety of aprotic solventssuch as THF, DMF, EtOAc, ether, benzene, toluene, or halogenatedsolvents such as chloroform or methylenechloride. THF is a preferredsolvent.

The reaction may be carried in the presence of an acid scavenger such astriethylamine, pyridine, or the like. Triethylamine is a preferred base.Additionally, an acylation catalyst such as 4-dimethylaminopyridine canbe used. The acylation reaction may be carried out under a variety ofreaction conditions from 0°-100° C. and under a nitrogen atmosphere.Usually, ambient temperature is sufficient. The reaction times can befrom 1-36 hours depending on the nature of the acylating moiety andother reaction conditions, progress of the reaction can be monitored bytechniques such as tlc. The resulting products are purified byevaporation of the reaction solvent in vacuo and re-dissolving theresidue, in EtOAc. The EtOAc solution is washed with aqueous base (1 NNaOH) and then with water and dried by filtration through anhydrous Na₂SO₄ or MgSO₄. The resulting organic solution is evaporated to a solid invacuo. The final product is then obtained by chromatographing the crudemixture on a silica gel column eluted with mixtures of EtOAc-hexane orthe like. A preferred solvent combination is 80% EtOAc-hexane. Theappropriate fractions containing the desired product may be identifiedby tlc and these fractions combined and evaporated to dryness in vacuo.

Mono- and di-esters of this invention may be prepared by using eitherone or two equivalents of the appropriate acylating reagent. The use ofone equivalent of acylating reagent gives rise to a statisticaldistribution of: dihydroxy (raloxifene, starting material), 4'hydroxy-6-acylraloxifene, 4'-acyl-6-hydroxyraloxifene, and4',6-diacylraloxifene. These compounds are easily separated bychromatographic procedures, silica gel eluted with mixtures of EtOAc andhexane. Thus, the various mono-derivatives may be obtained. Thelong-chain ester of formula I are tan, oily, and amorphous solids, orthick oils.

Mono- or di-glycerides, and 3-cholesterol derivatives of formula I maybe prepared by using a "linker" dicarboxylic acid. This moiety links thephenolic hydroxyls of the starting material (raloxifene) to thealcoholic hydroxyl of the mono- or di-glyceride or cholesterol viacarboxylic esters. Examples of such "linking" dicarboxylic acids areoxaloyl, succinoyl, glutaroyl, etc. The formation of such di-esters arewell known in the art.

Briefly, activated carboxylic acid moieties such as oxaloyl chloride,succinic anhydride, or glutaric anhydride can be used. Succinicanhydride is preferred.

In a manner similar to the formation of the acid esters described above,the mono- or preferred di-succinates of raloxifene are prepared. Thefree carboxylic acid moieties can further activated to react withalcoholic hydroxyls of cholesterol or mono- or di-glycerides. Theactivation of these free carboxylic acids may be accomplished byformation of mixed anhydrides with alkylchloroformates(i-butylchloroformate) and the intermediate mixed anhydride reacted withthe appropriate alcohol. Similarly, the free carboxyls may be directlyesterified with the appropriate alcohol using a dehydrating agent suchas DCC (di-cyclohexylcarbodimide) in an appropriate aprotic solvent suchas THF. Alternately, the "linking" dicarboxylic esters can be preparedby first, esterifying the lipophyllic alcohol, e.g., cholesterol, andthen forming the other ester bond with the phenol of raloxifene. Thefinal purified compounds of formula I can be obtained by chromatographictechniques. These compounds are oils or oily, amorphous solids.

Preferred embodiments of this invention are 4',6-distearoyl raloxifeneor([6-stearoyloxy-2-[4-(stearoyloxy)phenyl]benzo[b]thien-3-yl][4-(2-(1-piperidenyl)ethoxy)phenyl] methanone); 4',6-di-[3-cholesterolatesuccinoyl] raloxifene; and4',6-di-[1,2-di-stearoyl-3-glycerolsuccinate] raloxifene.

Below are described detailed preparations of selected compounds offormula I. These descriptions are for the purpose of illustration andare not meant to be limiting to the scope of this invention.

EXAMPLE 1[6-Stearoyloxy-2-[(4-stearoyloxy)phenyl]benzo[b]thien-3-yl][4-(2-(1-piperidenyl)ethoxy)phenyl]methanone(4',6-Distearoyl raloxifene)

A suspension of 2.6 g (0.005 mol) of raloxifene hydrochloride in 250 mLof THF was prepared. To this was added 11 g (0.1 mol) of triethylamineand 20 mg of dimethylaminopyridine (DMAP). The reaction mixture wasallowed to stir for 10 minutes at ambient temperature under anatmosphere of nitrogen. Stearoyl chloride, 3.3 g (0.011 mol), was addedand the reaction was allowed to proceed for sixteen hours. The reactionmixture was evaporated to dryness in vacuo and resuspended in 100 mL ofEtOAc. The EtOAc suspension was washed with water, then 1 N NaOH, andfinally with water. The organic layer was dried by filtration throughanhydrous Na₂ SO₄ and evaporated to dryness. The crude product wasfurther purified by chromatography on a silica gel column eluted withEtOAc. Evaporation of the desired fractions yielded 1.46 g of the titlecompound as a oily, low melting solid.

PMR: consistent with the proposed structure; MS: m/e=1006 FD; EA: Calc:C, 76.37; H, 9.51; N, 1.39 Found: C, 76.17; H, 9.56; N, 1.56; C₆₄ H₉₅NO₆ S.

EXAMPLE 2 4',6-Dilinolenoyl Raloxifene

This derivative was prepared in a manner similar to Example 1, using 2.6g (0.005 mol) of Raloxifene HCl, 2 g (0.02 mol) of triethylamine, 20 mgof DMAP, and 3.2 g (0.01 mol) of linolenoyl chloride in 250 mL of THF.The final product was chromatographed on a silica gel column eluted withEtOAc-hexane (8:2). This yielded 3.21 g of the title compound as clearoil.

PMR: consistent with the proposed structure; MS: m/e=994 FD.

EXAMPLE 3 4',6-Dilinoleoyl Raloxifene

The derivative was prepared in a manner similar to that in example 2,using 2.6 g (0.005 mol) of Raloxifene HCl, 3 g (0.03 mol) oftriethylamine, 20 mg of DMAP, and 3.2 g (0.01 mol) of linoleoyl chloridein 250 mL of THF. This yielded 4.76 g of the title compound as thickoil.

PMR: consistent with proposed structure; MS: m/e=998 FD

EXAMPLE 4 4',6-Dimyristoyl Raloxifene

This derivative was prepared in a manner similar to that in example 2,using 2.6 g (0.005 mol) of Raloxifene HCl, 5 g (0.05 mol) oftriethylamine, 20 mg of DMAP, and 2.8 g (0.012 mol) of myristoylchloride in 250 mL of THF. This yielded 2.61 g of the title compound asthick oil which solidified upon standing at room temperature.

PMR: consistent with proposed structure; MS: m/e-893 FD

EXAMPLE 5 4',6-Dipalmitoyl Raloxifene

This derivative was prepared in a manner similar to that in example 2,using 2.6 g (0.005 mol) of Raloxifene HCl, 5 g (0.05 mol) oftriethylamine, 20 mg of DMAP, and 3.3 g (0.012 mol) of palmitoylchloride in 250 mL of THF. This yielded 1.25 g of the title compound asa thick oil which solidified upon standing at room temperature.

PMR: consistent with proposed structure; MS: m/e=949 FD

The compounds used in the methods of this invention formpharmaceutically acceptable acid and base addition salts with a widevariety of organic and inorganic acids and bases and include thephysiologically acceptable salts which are often used in pharmaceuticalchemistry. Such salts are also part of this invention. Typical inorganicacids used to form such salts include hydrochloric, hydrobromic,hydroiodic, nitric, sulfuric, phosphoric, hypophosphoric and the like.Salts derived from organic acids, such as aliphatic mono anddicarboxylic acids, phenyl substituted alkanoic acids, hydroxyalkanoicand hydroxyalkandioic acids, aromatic acids, aliphatic and aromaticsulfonic acids, may also be used. Such pharmaceutically acceptable saltsthus include acetate, pharmaceutically acceptable salts thus includeacetate, phenylacetate, trifluoracetate, acrylate, ascorbate, benzoate,chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate,methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, bromide,isobutyrate, phenylbutyrate, β-hydroxybutyrate, butyne-1,4-dioate,hexyne-1,4-dioate, caprate, caprylate, chloride, cinnamate, citrate,formate, fumarate, glycollate, heptanoate, hippurate, lactate, malate,maleate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate,isonicotinate, nitrate, oxalate, phthalate, teraphthalate, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, propiolate, propionate, phenylpropionate, salicylate,sebacate, succinate, suberate, sulfate, bisulfate, pyrosulfate, sulfite,bisulfite, sulfonate, benzene-sulfonate, p-bromophenylsulfonate,chlorobenzenesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate,methane-sulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,p-toluenesulfonate, xylenesulfonate, tartarate, and the like. Apreferable salt is the hydrochloride salt.

The pharmaceutically acceptable acid addition salts are typically formedby reacting a compound of formula I with an equimolar or excess amountof acid. The reactants are generally combined in a mutual solvent suchas diethyl ether or benzene. The salt normally precipitates out ofsolution within about one hour to 10 days and can be isolated byfiltration or the solvent can be stripped off by conventional means.

Bases commonly used for formation of salts include ammonium hydroxideand alkali and alkaline earth metal hydroxides, carbonates andbicarbonates, as well as aliphatic and aromatic amines, aliphaticdiamines and hydroxy alkylamines. Bases especially useful in thepreparation of additional salts include ammonium hydroxide, potassiumcarbonate, sodium bicarbonate, calcium hydroxide, methylamine,diethylamine, ethylene diamine, cyclohexylamine and ethanolamine.

The pharmaceutically acceptable salts generally have enhanced solubilitycharacteristics compared to the compound from which they are derived,and thus are often more amenable to formulation as liquids or emulsions.

Pharmaceutical formulations can be prepared by procedures known in theart. For example, the compounds can be formulated with commonexcipients, diluents, or carriers, and formed into tablets, capsules,suspensions, powders, and the like. Examples of excipients, diluents,and carriers that are suitable for such formulations include thefollowing: fillers and extenders such as starch, sugars, mannitol, andsilicic derivatives; binding agents such as carboxymethyl cellulose andother cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; moisturizing agents such as glycerol; disintegrating agentssuch as agaragar, calcium carbonate, and sodium bicarbonate; agents forretarding dissolution such as paraffin; resorption accelerators such asquaternary ammonium compounds; surface active agents such as cetylalcohol, glycerol monostearate; adsorptive carriers such as kaolin andbentonite; and lubricants such as talc, calcium and magnesium stearate,and solid polyethyl glycols.

The compounds can also be formulated as elixirs or solutions forconvenient oral administration or as solutions appropriate for parentaladministration, for instance by intramuscular, subcutaneous orintravenous routes. Additionally, the compounds are well suited toformulation as sustained release dosage forms and the like. Theformulations can be so constituted that they release the activeingredient only or preferably in a particular part of the intestinaltract, possibly over a period of time. The coatings, envelopes, andprotective matrices may be made, for example, from polymeric substancesor waxes. alleviating post-menopausal syndrome in women which comprisesthe aforementioned method using compounds of Formula I and furthercomprises administering to a woman an effective amount of estrogen orprogestin. These treatments are particularly useful for treatingosteoporosis and lowering serum cholesterol because the patient willreceive the benefits of each pharmaceutical agent while the compounds ofthe present invention would inhibit undesirable side-effects of estrogenand progestin. Activity of these combination treatments in any of thepost-menopausal tests, infra, indicates that the combination treatmentsare useful for alleviating the symptoms of post-menopausal symptoms inwomen.

Various forms of estrogen and progestin are commercially available.Estrogen-based agents include, for example, ethynyl estrogen (0.01-0.03mg/day), mestranol (0.05-0.15 mg/day), and conjugated estrogenichormones such as Premarin® (Wyeth-Ayerst; 0.3-2.5 mg/day).Progestin-based agents include, for example, medroxyprogesterone such asProvera® (Upjohn; 2.5-10 mg/day), norethylnodrel (1.0-10.0 mg/day), andnonethindrone (0.5-2.0 mg/day). A preferred estrogen-based compound isPremarin, and norethylnodrel and norethindrone are preferredprogestin-based agents.

The method of administration of each estrogen- and progestin-based agentis consistent with that which is known in the art. For the majority ofthe methods of the present invention, compounds of Formula I areadministered continuously, from 1 to 3 times daily. However, cyclicaltherapy may especially be useful in the treatment of endometriosis ormay be used acutely during painful attacks of the disease. In the caseof restenosis, therapy may be limited to short (1-6 months) intervalsfollowing medical procedures such as angioplasty.

As used herein, the term "effective amount" means an amount of compoundof the present invention which is capable of alleviating the symptoms ofthe various pathological conditions herein described. The specific doseof a compound administered according to this invention will, of course,be determined by the particular circumstances surrounding the caseincluding, for example, the compound administered, the route ofadministration, the state of being of the patient, and the pathologicalcondition being treated. A typical daily dose will contain a nontoxicdosage level of from about 5 mg to about 600 mg/day of a compound of thepresent invention. Preferred daily doses generally will be from about 15mg to about 80 mg/day.

The local delivery of inhibitory amounts of active compound for thetreatment of restinosis can be by a variety of techniques whichadminister the compound at or near the proliferative site. Examples oflocal delivery techniques are not intended to be limiting but to beillustrative of the techniques available. Examples include localdelivery catheters, site specific carriers, implants, direct injection,or direct applications.

Local delivery by a catheter, including a permeable membrane catheter,allows the administration of a pharmaceutical agent directly to theproliferative lesion. Examples of local delivery using a ballooncatheter are described in EPO 383 492 A2 and U.S. Pat. No. 4,636,195(Wolinsky, Jan. 13, 1987).

Local delivery by an implant describes the surgical placement of amatrix that contains the pharmaceutical agent into the proliferativelesion. The implanted matrix releases the pharmaceutical agent bydiffusion, chemical reaction, or solvent activators. Lange, Science 249:1527-1533 (September, 1990).

An example of local delivery by an implant is the use of a stent. Stentsare designed to mechanically prevent the collapse and reocclusion of thecoronary arteries. Incorporating a pharmaceutical agent into the stentdelivers the drug directly to the proliferative site. Local delivery bythis technique is described in Kohn, Pharmaceutical Technology (October,1990).

Another example is a delivery system in which a polymer that containsthe pharmaceutical agent is injected into the lesion in liquid form. Thepolymer then cures to form the implant in situ. This technique isdescribed in PCT WO 90/03768 (Donn, Apr. 19, 1990).

Another example is the delivery of a pharmaceutical agent by polymericendoluminal sealing. This technique uses a catheter to apply a polymericimplant to the interior surface of the lumen. The pharmaceutical agentincorporated into the biodegradable polymer implant is thereby releasedat the surgical site. It is described in PCT WO 90/01969 (Schindler,Aug. 23, 1989).

A final example of local delivery by an implant is by direct injectionof vesicles or microparticulates into the proliferative site. Thesemicroparticulates may be composed of substances such as proteins,lipids, carbohydrates or synthetic polymers. These microparticulateshave the pharmaceutical agent incorporated throughout the microparticleor over the microparticule as a coating. Delivery systems incorporatingmicroparticulates are described in Lang, Science 249: 1527-1533(September, 1990) and Mathiowitz, et al., J. App. Poly. Sci., 26:809(1981).

Local delivery by site specific carriers describes attaching thepharmaceutical agent to a carrier which will direct the drug to theproliferative lesion. Examples of this delivery technique includes theuse of carriers such as a protein ligand or a monoclonal antibody.Lange, Science 249: 1527-1533 (September).

Local delivery by direct application includes the use of topicalapplications. An example of a local delivery by direct application isapplying the pharmaceutical agent directly to the arterial bypass graftduring the surgical procedure.

Another aspect of this invention are efficacious formulations of thecompounds of formula I for delivery into the highly lipophilicenvironment of the atherosclerotic plaque. Usual formulations of thetype used for intravenous injections (normally aqueous solutions) areinadvisable in light of the environment of the target site (plaque). Themajor considerations involving the formulation are 1) the formulatedproduct must pumped through the angioplasty catheter, 2) the formulatedproduct must facilitate the penetration of a compound of formula I intothe lipophyllic matrix of the plaque, and 3) the formulated product musthave minimal toxicity.

Carrier agents which would produce flowable solutions of a compound offormula I are DMSO, glycerol, liquid poly-alcohols, low molecular weightoils, and the like. These liquids may be adjusted with small amounts ofwater or alcohols to lower their viscosity. Additional agents such ascyclodextrins may be useful to aid the dissolution of the compounds offormula I in the carrier.

Penetration agents would facilitate entry into the plaque and includedetergents such as tritons, organophosphates, organosulfates,carboxymethylcellulose, DMSO, and the like.

Also, trace quantities of radio-contrasting agent or dye may beincorporated into the formulations to aid the attending physician toverify the effective delivery of the formulation to its intended targetsite.

The exact amount of a compound of formula I and the volume of theformulated product for use in inhibiting atherosclerotic plaque at thelocal site may vary depending the circumstances and is best determinedby the attending physician. Such factors as the depth and size of theatherosclerotic plaque to be treated are highly variable, in general,0.5-2.0 mg would be an effective amount of a compound of formula I andthis to be delivered in a volume of 1-3 mL. Thus various strengths andvolumes of these formulations would be necessary to allow the greatestlatitude of choice to the attending physician.

The clinical use of this invention would not differ greatly from thestandard angioplastic procedure currently in practice. An additionalbenefit of this invention, due to the inclusion of a radio-contrastingagent in the formulated product, is that the attending physician mayverify the location and the extent of penetration of the formulationinto the plaque and surrounding tissue by radiographic techniques.

Listed below are formulations for the compounds of formula I. Theseformulations are given for purposes of illustration and are not intendedto limit the scope of this invention in anyway. The term "activeingredient" means a compound of formula I.

    ______________________________________                                        Formulation 1                                                                 Ingredient         Quantity (mg/capsule)                                      ______________________________________                                        Active Ingredient  0.5-3.0 mg                                                 B-cyclodextrin     0.1 mg                                                     DMSO               1.5 mL                                                     Barium Oxide       0.1 mg                                                     Sterile Water                                                                 ______________________________________                                    

A compound of formula I (0.5-3.0 mg) and 0.1 mg of B-cyclodextrin isdissolved in 1.5 mL of DMSO and 0.1 mg of barium oxide is added. Themixture is heated to induce solution (50° C.) and allowed to cool toambient temperature. Sterile water is added to bring the volume to 2 mL.

    ______________________________________                                        Formulation 2                                                                 Ingredient         Quantity (mg/capsule)                                      ______________________________________                                        Active Ingredient  0.5-3.0 mg                                                 glycerol           1 mL                                                       DMSO               1 mL                                                       Triton X           0.1 mg                                                     Barium Oxide       0.1 mg                                                     ______________________________________                                    

A compound of formula I (0.5-3.0 mg) is dissolved in 1 mL of DMSO.Triton X (0.1 mg) and barium oxide (0.1 mg) are added along with 1 mL ofglycerol. The mixture is thoroughly mixed.

    ______________________________________                                        Formulation 3: Gelatin Capsules                                               Hard gelatin capsules are prepared using the following:                       Ingredient          Quantity (mg/capsule)                                     ______________________________________                                        Active ingredient   0.1-1000                                                  Starch, NF          0-650                                                     Starch flowable powder                                                                            0-650                                                     Silicone fluid 350 centistokes                                                                    0-15                                                      ______________________________________                                    

The formulation above may be changed in compliance with the reasonablevariations provided.

A tablet formulation is prepared using the ingredients below:

    ______________________________________                                        Formulation 4: Tablets                                                        Ingredient           Quantity (mg/tablet)                                     ______________________________________                                        Active ingredient    2.5-1000                                                 Cellulose, microcrystalline                                                                        200-650                                                  Silicon dioxide, fumed                                                                             10-650                                                   Stearate acid        5-15                                                     ______________________________________                                    

The components are blended and compressed to form tablets.

Alternatively, tablets each containing 2.5-1000 mg of active ingredientare made up as follows:

    ______________________________________                                        Formulation 5: Tablets                                                        Ingredient           Quantity (mg/tablet)                                     ______________________________________                                        Active ingredient    25-1000                                                  Starch               45                                                       Cellulose, microcrystalline                                                                        35                                                       Polyvinylpyrrolidone 4                                                        (as 10% solution in water)                                                    Sodium carboxymethyl cellulose                                                                     4.5                                                      Magnesium stearate   0.5                                                      Talc                 1                                                        ______________________________________                                    

The active ingredient, starch, and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders which are thenpassed through a No. 14 mesh U.S. sieve. The granules so produced aredried at 50°-60° C. and passed through a No. 18 mesh U.S. sieve. Thesodium carboxymethyl starch, magnesium stearate, and talc, previouslypassed through a No. 60 U.S. sieve, are then added to the granuleswhich, after mixing, are compressed on a tablet machine to yieldtablets.

Suspensions each containing 0.1-1000 mg of medicament per 5 ml dose aremade as follows:

    ______________________________________                                        Formulation 6: Suspensions                                                    Ingredient           Quantity (mg/5 ml)                                       ______________________________________                                        Active ingredient    0.1-1000 mg                                              Sodium carboxymethyl cellulose                                                                     50 mg                                                    Syrup                1.25 mg                                                  Benzoic acid solution                                                                              0.10 mL                                                  Flavor               q.v.                                                     Color                q.v.                                                     Purified water to    5 mL                                                     ______________________________________                                    

The medicament is passed through a No. 45 mesh U.S. sieve and mixed withthe sodium carboxymethyl cellulose and syrup to form a smooth paste. Thebenzoic acid solution, flavor, and color are diluted with some of thewater and added, with stirring. Sufficient water is then added toproduce the required volume.

An aerosol solution is prepared containing the following ingredients:

    ______________________________________                                        Formulation 7: Aerosol                                                        Ingredient           Quantity (% by weight)                                   ______________________________________                                        Active ingredient    0.25                                                     Ethanol              25.75                                                    Propellant 22 (Chlorodifluoromethane)                                                              70.00                                                    ______________________________________                                    

The active ingredient is mixed with ethanol and the mixture added to aportion of the propellant 22, cooled to 30° C., and transferred to afilling device. The required amount is then fed to a stainless steelcontainer and diluted with the remaining propellant. The valve units arethen fitted to the container.

Suppositories are prepared as follows:

    ______________________________________                                        Formulation 8: Suppositories                                                  Ingredient       Quantity (mg/suppository)                                    ______________________________________                                        Active ingredient                                                                              250                                                          Saturated fatty acid glycerides                                                                2,000                                                        ______________________________________                                    

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimal necessary heat. The mixture is then poured into asuppository mold of nominal 2 g capacity and allowed to cool.

An intravenous formulation is prepared as follows:

    ______________________________________                                        Formulation 9: Intravenous Solution                                           Ingredient            Quantity                                                ______________________________________                                        Active ingredient     50 mg                                                   Isotonic saline       1,000 mL                                                ______________________________________                                    

The solution of the above ingredients is intravenously administered to apatient at a rate of about 1 mL per minute.

    ______________________________________                                        Formulation 10: Combination Capsule I                                         Ingredient    Quantity (mg/capsule)                                           ______________________________________                                        Active ingredient                                                                           50                                                              Premarin      1                                                               Avicel pH 101 50                                                              Starch 1500   117.50                                                          Silicon Oil   2                                                               Tween 80      0.50                                                            Cab-O-Sil     0.25                                                            ______________________________________                                    

    ______________________________________                                        Formulation 11: Combination Capsule II                                        Ingredient    Quantity (mg/capsule)                                           ______________________________________                                        Active ingredient                                                                           50                                                              Norethylnodrel                                                                              5                                                               Avicel pH 101 82.50                                                           Starch 1500   90                                                              Silicon Oil   2                                                               Tween 80      0.50                                                            ______________________________________                                    

    ______________________________________                                        Formulation 12: Combination Tablet                                            Ingredient     Quantity (mg/capsule)                                          ______________________________________                                        Active ingredient                                                                            50                                                             Premarin       1                                                              Corn Starch NF 50                                                             Povidone, K29-32                                                                             6                                                              Avicel pH 101  41.50                                                          Avicel pH 102  136.50                                                         Crospovidone XL10                                                                            2.50                                                           Magnesium Stearate                                                                           0.50                                                           Cab-O-Sil      0.50                                                           ______________________________________                                    

More generally, the total active ingredients in such formulationscomprises from 0.1% to 99.9% by weight of the formulation. By"pharmaceutically acceptable" it is meant the carrier, diluent,excipients and salt must be compatible with the other ingredients of theformulation, and not deleterious to the recipient thereof.

As mentioned previously, one of the novel aspects of this invention isthe enhanced lipophillicity of compounds of formula I. Experimentaldemonstration of this enhanced lipophillicity, and thus enhanced abilityto penetrate an atherosclerotic plaque, may be shown by severaltechniques: 1) excellent solubility of the compounds of formula I inlipophillic solvents such as EtOAc, 2) excellent lipophillic characterof the compounds of formula I as shown by a standard, chemical techniquesuch as log_(p) partion coefficients (n-octanol/water), or 3) enhanceddiffusion rates of the compounds of formula I into a lipophillic matrixsuch as cholesterol.

The following assays are used to illustrate the invention:

Balloon Injury of Carotid Arteries

Balloon injury to the left common carotid arteries of male,Sprague-Dawley rats (350-400 g) is accomplished by three passes of aninflated 2F Fogarty balloon catheter (Baxter Healthcare, McGraw Park,Ill.) as described by Clowes A. W., et al., Lab. Invest. 49, p.208-215(1983). Animals are anesthetized with Ketamine (80 mg/kg, IM) and Rompun(16 mg/kg, IM). Entry of the balloon catheter to the left common carotidartery is made by a nick in the external carotid artery, which is tiedoff at the end of the surgical procedure.

Following balloon injury, a single daily dose of a compound of formula Iis applied to the exterior of the injured carotid artery as a "loadingdose" of drug in a small volume (120 uL). Subsequent to this, continuousdelivery of the compound to the adventitial (exterior) space surroundingthe injured carotid artery is accomplished by means of a miniosmoticpump (Alzet, 2ML2, Palo Alto, Calif.) implanted subcutaneously in theback of the rat. Pumps are primed before surgery and implantedimmediately following balloon injury. Dosing solutions are delivered tothe adventitial space via a micro-renathane catheter (MRE-40). Thecatheter is sutured in place with two ligatures (4-0 silk) to the leftexternal carotid artery, and the tip is positioned to deliver the drugsolutions at the midpoint of the common carotid artery. The dosingvehicle employed in this study is 20% cyclodextrin in sterile water.

Fourteen days post surgery, animals are anesthetized (vide supra) andperfused through the abdominal aorta in a retrograde manner atphysiological pressure with a zinc formalin fixative (Anatech LTD,Battle Creek, Mich.). Middle sections (5 mm) of the carotids are removedfrom the animals, processed, and embedded in paraffin. Three adjacentcross sections (5 μm thick) of each vessel are cut, stained withhematoxylin and eosin, and cross sectional intimal areas are quantitatedwith an image analyzer (Quantimat 970, Cambridge Inst. Cambridge. UK).

The results of this experiment demonstrate the ability of the compoundsof formula I to inhibit the reduction of intimal area due to restenosisinitiated by injury of the balloon catheter.

In the examples illustrating the methods, a post-menopausal model isused in which effects of different treatments upon circulating lipidsare determined.

Seventy-five day old female Sprague Dawley rats (weight range of 200 to225g) are obtained from Charles River Laboratories (Portage, Mich.). Theanimals are either bilaterally ovariectomized (OVX) or exposed to a Shamsurgical procedure at Charles River Laboratories, and then shipped afterone week. Upon arrival, they are housed in metal hanging cages in groupsof 3 or 4 per cage and had ad libitum access to food (calcium contentapproximately 0.5%) and water for one week. Room temperature ismaintained at 22.2°±1.70° C. with a minimum relative humidity of 40%.The photoperiod in the room is 12 hours light and 12 hours dark.

Dosing Regimen Tissue Collection

After a one week acclimation period (therefore, two weeks post-OVX)daily dosing with test compound is initiated. 17α-ethynyl estradiol orthe test compound are given orally, unless otherwise stated, as asuspension in 1% carboxymethylcellulose or dissolved in 20%cyclodextrin. Animals are dosed daily for 4 days. Following the dosingregimen, animals are weighed and anesthetized with a ketamine: xylazine(2:1, V:V) mixture and a blood sample is collected by cardiac puncture.The animals are then sacrificed by asphyxiation with CO₂, the uterus isremoved through a midline incision, and a wet uterine weight isdetermined.

Cholesterol Analysis

Blood samples are allowed to clot at room temperature for 2 hours, andserum is obtained following centrifugation for 10 minutes at 3000 rpm.Serum cholesterol is determined using a Boehringer Mannheim Diagnosticshigh performance cholesterol assay. Briefly the cholesterol is oxidizedto cholest-4-en-3-one and hydrogen peroxide. The hydrogen peroxide isthen reacted with phenol and 4-aminophenazone in the presence ofperoxidase to produce a p-quinone imine dye, which is readspectrophotemetrically at 500 nm. Cholesterol concentration is thencalculated against a standard curve. The entire assay is automated usinga Biomek Automated Workstation.

Uterine Eosinophil Peroxidase (EPO) Assay

Uteri are kept at 4° C. until time of enzymatic analysis. The uteri arethen homogenized in 50 volumes of 50 mM Tris buffer (pH-8.0) containing0.005% Triton X-100. Upon addition of 0.01% hydrogen peroxide and 10 mMO-phenylenediamine (final concentrations) in Tris buffer, increase inabsorbance is monitored for one minute at 450 nm. The presence ofeosonophils in the uterus is an indication of estrogenic activity of acompound. The maximal velocity of a 15 second interval is determinedover the initial, linear portion of the reaction curve.

Source of Compound

17α-ethynyl estradiol is obtained from Sigma Chemical Co., St. Louis,Mo.

Osteoporosis Test Procedure

Following the General Preparation Procedure, infra, the rats are treateddaily for 35 days (6 rats per treatment group) and sacrificed by carbondioxide asphyxiation on the 36th day. The 35 day time period issufficient to allow maximal reduction in bone density, measured asdescribed herein. At the time of sacrifice, the uteri are removed,dissected free of extraneous tissue, and the fluid contents are expelledbefore determination of wet weight in order to confirm estrogendeficiency associated with complete ovariectomy. Uterine weight isroutinely reduced about 75% in response to ovariectomy. The uteri arethen placed in 10% neutral buffered formalin to allow for subsequenthistological analysis.

The right femurs are excised and digitilized x-rays generated andanalyzed by an image analysis program (NIH image) at the distalmetaphysis. The proximal aspect of the tibiae from these animals arealso scanned by quantitative computed tomography.

In accordance with the above procedures, compounds of the presentinvention and ethynyl estradiol (EE₂) in 20% hydroxypropylβ-cyclodextrin are orally administered to test animals.

MCF-7 Proliferation Assay

MCF-7 breast adenocarcinoma cells (ATCC HTB 22) are maintained in MEM(minimal essential medium, phenol red-free, Sigma, St. Louis, Mo.)supplemented with 10% fetal bovine serum (FBS) (V/V), L-glutamine (2mM), sodium pyruvate (1 mM), HEPES{(N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]10 mM},non-essential amino acids and bovine insulin (1 ug/mL) (maintenancemedium). Ten days prior to assay, MCF-7 cells are switched tomaintenance medium supplemented with 10% dextran coated charcoalstripped fetal bovine serum (DCC-FBS) assay medium) in place of 10% FBSto deplete internal stores of steroids. MCF-7 cells are removed frommaintenance flasks using cell dissociation medium (Ca++/Mg++ free HBSS(phenol red-free) supplemented with 10 mM HEPES and 2 mM EDTA). Cellsare washed twice with assay medium and adjusted to 80,000 cells/mL.Approximately 100 μL (8,000 cells) are added to flat-bottom microculturewells (Costar 3596) and incubated at 37° C. in a 5% CO₂ humidifiedincubator for 48 hours to allow for cell adherence and equilibrationafter transfer. Serial dilutions of drugs or DMSO as a diluent controlare prepared in assay medium and 50 μL transferred to triplicatemicrocultures followed by 50 μL assay medium for a final volume of 200μL. After an additional 48 hours at 37° C. in a 5% CO₂ humidifiedincubator, microcultures are pulsed with tritiated thymidine (1uCi/well) for 4 hours. Cultures are terminated by freezing at -70° C.for 24 hours followed by thawing and harvesting of microcultures using aSkatron Semiautomatic Cell Harvester. Samples are counted by liquidscintillation using a Wallac BetaPlace βcounter.

DMBA-Induced Mammary Tumor Inhibition

Estrogen-dependent mammary tumors are produced in female Sprague-Dawleyrats which are purchased from Harlan Industries, Indianapolis, Ind. Atabout 55 days of age, the rats receive a single oral feeding of 20 mg of7,12-dimethylbenz[a]anthracene (DMBA). About 6 weeks after DMBAadministration, the mammary glands are palpated at weekly intervals forthe appearance of tumors. Whenever one or more tumors appear, thelongest and shortest diameters of each tumor are measured with a metriccaliper, the measurements are recorded, and that animal is selected forexperimentation. An attempt is made to uniformly distribute the varioussizes of tumors in the treated and control groups such thataverage-sized tumors are equivalently distributed between test groups.Control groups and test groups for each experiment contain 5 to 9animals.

Compounds of Formula I are administered either through intraperitonealinjections in 2% acacia, or orally. Orally administered compounds areeither dissolved or suspended in 0.2 mL corn oil. Each treatment,including acacia and corn oil control treatments, is administered oncedaily to each test animal. Following the initial tumor measurement andselection of test animals, tumors are measured each week by theabove-mentioned method. The treatment and measurements of animalscontinue for 3 to 5 weeks at which time the final areas of the tumorsare determined. For each compound and control treatment, the change inthe mean tumor area is determined.

Uterine Fibrosis Test Procedures

Assay 1

Between 3 and 20 women having uterine fibrosis are administered acompound of the present invention. The amount of compound administeredis from 0.1 to 1000 mg/day, and the period of administration is 3months.

The women are observed during the period of administration, and up to 3months after discontinuance of administration, for effects on uterinefibrosis.

Assay 2

The same procedure is used as in Test 1, except the period ofadministration is 6 months.

Assay 3

The same procedure is used as in Test 1, except the period ofadministration is 1 year.

Assay 4

A. Induction of fibroid tumors in guinea pig.

Prolonged estrogen stimulation is used to induce leiomyomata in sexuallymature female guinea pigs. Animals are dosed with estradiol 3-5 timesper week by injection for 2-4 months or until tumors arise. Treatmentsconsisting of a compound of the invention or vehicle is administereddaily for 3-16 weeks and then animals are sacrificed and the uteriharvested and analyzed for tumor regression.

B. Implantation of human uterine fibroid tissue in nude mice.

Tissue from human leiomyomas are implanted into the peritoneal cavityand or uterine myometrium of sexually mature, castrated, female, nudemice. Exogenous estrogen are supplied to induce growth of the explantedtissue. In some cases, the harvested tumor cells are cultured in vitroprior to implantation. Treatment consisting of a compound of the presentinvention or vehicle is supplied by gastric lavage on a daily basis for3-16 weeks and implants are removed and measured for growth orregression. At the time of sacrifice, the uteri is harvested to assessthe status of the organ.

Assay 5

A. Tissue from human uterine fibroid tumors is harvested and maintained,in vitro, as primary nontransformed cultures. Surgical specimens arepushed through a sterile mesh or sieve, or alternately teased apart fromsurrounding tissue to produce a single cell suspension. Cells aremaintained in media containing 10% serum and antibiotic. Rates of growthin the presence and absence of estrogen are determined. Cells areassayed for their ability to produce complement component C3 and theirresponse to growth factors and growth hormone. In vitro cultures areassessed for their proliferative response following treatment withprogestins, GnRH, a compound of the present invention and vehicle.Levels of steroid hormone receptors are assessed weekly to determinewhether important cell characteristics are maintained in vitro. Tissuefrom 5-25 patients are utilized.

Activity in at least one of the above tests indicates the compounds ofthe present invention are of potential in the treatment of uterinefibrosis.

Endometriosis Test Procedure

In Tests 1 and 2, effects of 14-day and 21-day administration ofcompounds of the present invention on the growth of explantedendometrial tissue can be examined.

Assay 1

Twelve to thirty adult CD strain female rats are used as test animals.They are divided into three groups of equal numbers. The estrous cycleof all animals is monitored. On the day of proestrus, surgery isperformed on each female. Females in each group have the left uterinehorn removed, sectioned into small squares, and the squares are looselysutured at various sites adjacent to the mesenteric blood flow. Inaddition, females in Group 2 have the ovaries removed.

On the day following surgery, animals in Groups 1 and 2 receiveintraperitoneal injections of water for 14 days whereas animals in Group3 receive intraperitoneal injections of 1.0 mg of a compound of thepresent invention per kilogram of body weight for the same duration.Following 14 days of treatment, each female is sacrificed and theendometrial explants, adrenals, remaining uterus, and ovaries, whereapplicable, are removed and prepared for histological examination. Theovaries and adrenals are weighed.

Assay 2

Twelve to thirty adult CD strain female rats are used as test animals.They are divided into two equal groups. The estrous cycle of all animalsis monitored. On the day of proestrus, surgery is performed on eachfemale. Females in each group have the left uterine horn removed,sectioned into small squares, and the squares are loosely sutured atvarious sites adjacent to the mesenteric blood flow.

Approximately 50 days following surgery, animals assigned to Group 1receive intraperitoneal injections of water for 21 days whereas animalsin Group 2 receive intraperitoneal injections of 1.0 mg of a compound ofthe present invention per kilogram of body weight for the same duration.Following 21 days of treatment, each female is sacrificed and theendometrial explants and adrenals are removed and weighed. The explantsare measured as an indication of growth. Estrous cycles are monitored.

Assay 3

A. Surgical induction of endometriosis

Autographs of endometrial tissue are used to induce endometriosis inrats and/or rabbits. Female animals at reproductive maturity undergobilateral oophorectomy, and estrogen is supplied exogenously thusproviding a specific and constant level of hormone. Autologousendometrial tissue is implanted in the peritoneum of 5-150 animals andestrogen supplied to induce growth of the explanted tissue. Treatmentconsisting of a compound of the present invention is supplied by gastriclavage on a daily basis for 3-16 weeks, and implants are removed andmeasured for growth or regression. At the time of sacrifice, the intacthorn of the uterus is harvested to assess status of endometrium.

B. Implantation of human endometrial tissue in nude mice.

Tissue from human endometrial lesions is implanted into the peritoneumof sexually mature, castrated, female, nude mice. Exogenous estrogen issupplied to induce growth of the explanted tissue. In some cases, theharvested endometrial cells are cultured in vitro prior to implantation.Treatment consisting of a compound of the present invention supplied bygastric lavage on a daily basis for 3-16 weeks, and implants are removedand measured for growth or regression. At the time of sacrifice, theuteri is harvested to assess the status of the intact endometrium.

Assay 4

A. Tissue from human endometrial lesions is harvested and maintained invitro as primary nontransformed cultures. Surgical specimens are pushedthrough a sterile mesh or sieve, or alternately teased apart fromsurrounding tissue to produce a single cell suspension. Cells aremaintained in media containing 10% serum and antibiotic. Rates of growthin the presence and absence of estrogen are determined. Cells areassayed for their ability to produce complement component C3 and theirresponse to growth factors and growth hormone. In vitro cultures areassessed for their proliferative response following treatment withprogestins, GnRH, a compound of the invention, and vehicle. Levels ofsteroid hormone receptors are assessed weekly to determine whetherimportant cell characteristics are maintained in vitro. Tissue from 5-25patients is utilized.

Activity in any of the above assays indicates that the compounds of thepresent invention are useful in the treatment of endometriosis.

Inhibition of Aortal Smooth Cell Proliferation/Restenosis Test Procedure

Compounds of the present invention have capacity to inhibit aortalsmooth cell proliferation. This can be demonstrated by using culturedsmooth cells derived from rabbit aorta, proliferation being determinedby the measurement of DNA synthesis. Cells are obtained by explantmethod as described in Ross, J. of Cell Bio. 50: 172 (1971). Cells areplated in 96 well microtiter plates for five days. The cultures becomeconfluent and growth arrested. The cells are then transferred toDulbecco's Modified Eagle's Medium (DMEM) containing 0.5-2% plateletpoor plasma, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg mlstreptomycin, 1 mC/ml ³ H-thymidine, 20 ng/ml platelet-derived growthfactor, and varying concentrations of the present compounds. Stocksolution of the compounds is prepared in dimethyl sulphoxide and thendiluted to appropriate concentration (0.01-30 mM) in the above assaymedium. Cells are then incubated at 37° C. for 24 hours under 5% CO₂/95% air. At the end of 24 hours, the cells are fixed in methanol. ³ Hthymidine incorporation in DNA is then determined by scintillationcounting as described in Bonin, et al., Exp. Cell Res. 181: 475-482(1989).

Inhibition of aortal smooth muscle cell proliferation by the compoundsof the present invention are further demonstrated by determining theireffects on exponentially growing cells. Smooth muscle cells from rabbitaortae are seeded in 12 well tissue culture plates in DMEM containing10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100mg/ml streptomycin. After 24 hours, the cells are attached and themedium is replaced with DMEM containing 10% serum, 2 mM L-glutamine, 100U/ml penicillin, 100 mg/ml streptomycin, and desired concentrations ofthe compounds. Cells are allowed to grow for four days. Cells aretreated with trypsin and the number of cells in each culture isdetermined by counting using a ZM-Coulter counter.

Activity in the above assays indicates that the compounds of the presentinvention are of potential in the treatment of restenosis.

We claim:
 1. A method for inhibiting estrogen-dependent cancercomprising administering to a human in need thereof a compound offormula (I): ##STR3## wherein: R₁ is N-pyrrolidinyl or N-piperidinyl;R₂and R₃ are independently hydrogen, --CO--(C₁₀ -C₂₂ alkyl), --CO--(C₁₀-C₂₂ branched alkyl), --CO--(C₁₀ -C₂₂ alkenyl), --CO--(C₁₀ -C₂₂polyalkenyl), --CO--(C₁₀ -C₂₂ alkynyl), or --CO--(CH₂)_(n) COR₄ ;provided that when R₁ is N-piperidinyl, neither R₂ or R₃ is --CO--(C₁₀-C₂₂ alkyl) or --CO--(C₁₀ -C₂₂ branched alkyl); and provided one of R₂or R₃ is not hydrogen; R₄ is -3-cholesteryl or --O(CH₂)₂ (OR₅)CH₂ OR₆ ;R₅ and R₆ are independently hydrogen, --CO--(C₁₀ -C₂₂ alkyl), --CO--(C₁₀-C₂₂ branched alkyl), --CO--(C₁₀ -C₂₂ alkenyl), --CO--(C₁₀ -C₂₂polyalkenyl), or --CO--(C₁₀ -C₂₂ alkynyl); provided one of R₅ or R₆ isnot hydrogen; and n is 0-4; or a pharmaceutically acceptable salt orsolvate thereof.
 2. The method of claim 1 wherein R₁ is N-piperidinyland R₂ and R₃ are each stearoyl.
 3. The method of claim 1 wherein R₁ isN-piperidinyl and R₂ and R₃ are each 3-cholesterylsuccinoyl.
 4. Themethod of claim 1 wherein R₁ is N-piperidinyl and R₂ and R₃ are each1,2-di-stearoylglycerol-3-succinoyl.
 5. The method of claim 1 where saidcompound is selected from the group consisting of4',6-distearoylraloxifene, 4',6-di-[3-cholesterolatesuccinoyl]raloxifene, or 4',6-di-[1,2-di-stearoyl-3-glycerolsuccinate]raloxifene.
 6. The method of claim 1 where theestrogen-dependent cancer is selected from the group consisting ofbreast and uterine cancer.